1. Field of the Invention
The present invention concerns new pharmaceutical compositions intended to prevent and/or treat the expression of brooding behavior in birds, and more particularly in the turkey. The invention also relates to the procedures for the preparation of these pharmaceutical compositions.
2. Background of the Related Art
The brooding or incubation period is one of the major phases in the natural reproductive cycle of birds. However, the aptitude for brooding, far from being considered a quality of breeders, is instead an unfavorable factor, inasmuch as all of the eggs to be hatched are incubated artificially. Brooding behavior still persists among several species of domestic birds, particularly among chickens, ducks, and turkeys. This behavior leads to a notable reduction in the number of incubatable eggs and thus to a very significant loss of income for the breeders.
The observation and understanding of this behavior have made it possible to develop the first ways to combat broodiness on breeding farms.
In the turkeys on a breeding farm, the first brooders are identified after the peak of production, i.e., toward the fourth week of egg-laying. In the absence of preventive actions, their number reaches a maximum toward the eighth week [1] [2] [3]. The passage from layer to brooder status is normally associated with a cessation of laying and with regression of the reproductive apparatus (ovary and oviduct). Certain morphological changes (such as narrowing of the pelvic bones and development of incubatory plates) and behavioral changes (an increase in the amount of time spent on the nest, defense of the nest, ruffled plumage, vocalizations, reduced feeding activity) can then be observed. However, the simultaneous appearance of the entire group of these changes is unusual in the turkey. Consequently, early identification of brooders is a trickier matter in this species.
The hypothesis of humoral control of the expression of brooding was advanced as early as 1927 [4]. Because an elevation of plasma concentrations of prolactin was associated with the expression of this behavior, this hormone was designated as the causal factor [5]; but this has never been formally confirmed. Recent studies using radioimmunoassay methods have confirmed that the plasma concentrations of LH [i.e., luteinizing hormone], as well as the concentrations of the ovarian steroids estradiol and progesterone, are minimal during brooding, while the plasma level of prolactin is maximal [6].
Nevertheless, it has been possible to detect several factors that affect the expression of brooding behavior.
Genetic factors were identified after it was observed that the percentage of turkeys capable of externalizing brooding behavior in a given a population varied as a function of the strain. The expression of brooding, as a quantitative characteristic, is controlled by several genes, and its transmission simultaneously involves both sex-linked factors and autosomal factors [7] [8]. An estimate indicates that the inheritability of this characteristic in the turkey is between 0.30 and 0.40 [9].
The incidence of brooding also depends on exogenous factors associated with the environment and on endogenous factors associated with the physiological condition of the birds. The most frequently cited environmental factors are the raising method, temperature, and light. The centralized integration of these exogenous stimuli and endogenous stimuli plays a determining role in the emergence of this behavior. One or more monoamines (such as dopamine or serotonin) and hypothalamic peptides (such as TRH [thyrotropin-releasing hormone] or VIP [vasoactive intestinal polypeptide]) may also be involved in the chain of mechanisms leading to brooding [10] [11]. It has also been suggested that ovarian steroids encourage the expression of brooding by stimulating the secretion of prolactin during the passage from layer status to brooder status.
On the basis of these studies, three ways of combating brooding have been developed, i.e.:
Selection, PA1 Physical procedures; and PA1 Drug treatments.
The implementation of rigorous selection programs, applied in conjunction with strict raising techniques, has made it possible to reduce the percentage of turkeys that express brooding on breeding farms. However, depending on the strains, 10 to 70 percent of the turkeys in a flock still express this behavior during the reproductive cycle. As a result, bird breeders are required to implement appropriate preventive and curative measures.
The physical procedures can be implemented both preventively and curatively.
Preventively speaking, the inhibitory properties of a change in the environment with regard to the expression of brooding [13] [14] [15] [16] are known. These properties can be utilized define standard raising procedures that minimize the expression of brooding. Therefore, it is desirable not to exceed a maximum density of 2 turkeys per square meter, and to have available a sufficient number of nests (e.g., one for every 5 turkeys), which will limit the act of laying on the ground, which also encourages brooding. It is also important to provide uniform lighting in the poultry house, to control the temperature (particularly by providing well-ventilated premises), to collect the eggs often (10 to 20 times a day), to remove the turkeys from the nests during collection, and to prevent access to the nests at night. For this purpose, nests are available that are equipped with a system that automatically ejects the birds and collects the eggs, thereby reducing the breeder's workload. However, these standard procedures do not completely prevent the expression of brooding.
The curative treatments utilized by the professionals are based on modifications of the breeding environment. These modifications include the use of high-intensity lights, audible signals, and/or electrical stimuli. However, the most common modification consists of placing the brooding turkeys in a foreign environment, known as the "de-brooding cage." The group version of this cage has a lattice-work floor or a floor that is littered with sand, while the individual cages constitute the most frequently used variant of this approach. A turkey generally stays in the cage for 3 to 6 days and, in all cases, is deprived of a nest in the cage.
This type of treatment induces a reduction of prolactinemia and is therefore probably effective for preventing the expression of brooding behavior over the short term [3]. Conversely, it probably does not always have the effect hoped for by breeders, particularly among turkeys that still lay after they have been identified as brooders and therefore are subjected to the treatment. The treatment of a turkey at this particular stage will induce in her an important mobilization of the bodily reserves that subsequently cause a suspension of laying in some of these birds. Therefore, this type of treatment entails the risk of blocking the actual reproductive capabilities of the turkeys that are potentially the most productive. Therefore, the brooding turkeys must be identified with the greatest care, in order to prevent the unintentional treatment of non-brooding turkeys, particularly at the beginning of the production period.
In short, these physical treatments are not truly satisfactory, and also require the installation of special facilities, at a significant cost to the breeder.
In parallel with these physical procedures, pharmacological treatments to combat brooding have also been proposed. The drugs proposed are essentially of three types, i.e., those that have a steroidogenic effect, those that stimulate the secretion of LH, and those that inhibit the secretion of prolactin.
The first tests of pharmacological treatments, consisting of the administration of steroids or of steroid analogs, had the goal of compensating for the decrease in circulatory levels of the steroids associated with brooding. This type of treatment most often includes an interruption of brooding, but also tends to delay the return to laying, due to the negative retro-control that is exerted by the steroids on the secretion of LH. Therefore, this type of drug was not selected.
The goal of treatment with drugs that stimulate the secretion of LH is to compensate for the decrease in LH that occurs during brooding. It has been shown that clomiphene citrate (an antiestrogenic substance) is active in interrupting brooding and stimulating the return to laying [17]. However, contradictory results for the efficacy of this substance have been reported by several authors [18] [1]. In particular, it has been shown that there is a decrease in the levels of LH after treatment--a development which is incompatible with a rapid return to laying [20]. It has also been observed that LHRH [luteinizing-hormone releasing hormone] is active in stimulating the secretion of LH in brooding turkeys [21] [22] [23]. However, the repeated injection of LHRH induces a desensitization of the pituitary, and does not stimulate ovarian activity in the dwarf hen [18]. Conversely, the injection of PMSG [pregnant-mare serum gonadotropin] stimulates ovarian activity in brooding turkeys, but does not induce either the interruption of brooding or a return to laying [24].
The inhibitory or stimulatory effects of several monoamines (e.g. serotonin or dopamine) and hypothalamic peptides (TRH, VIP) on the secretion of prolactin have been demonstrated in the turkey [10] [11] [25], and the utilization of substances that interfere with the synthesis, release, and/or action mechanism of these substances in order to control brooding has been the subject of various experiments. Bromocriptine, a dopamine analog that inhibits the secretion of prolactin in mammals, encourages the expression of brooding in the turkey [17]. Conversely, pimozide, which blocks the dopaminergic receptors, is effective in interrupting brooding and stimulating the return to laying [26]. However, this result cannot be obtained unless the turkey is treated during the intermediate phase between laying and brooding, with the associated implication of early identification. It has also been observed that the secretion of prolactin is stimulated by serotonin [27] [11] and by PCPA (a serotonin-synthesis inhibitor), and eventually induces eventually the interruption of brooding [28] [29]. On the other hand, the positive results that have been reported for the restoration of ovarian activity [28] have not yet been confirmed.
It has furthermore been shown that the intracerebral administration of prolactin ovine retards the entry into production of immature turkeys, and also induces nest-making behavior in laying turkeys, with an associated decrease in plasma concentrations of prolactin [30].
The most recent approaches consist of utilizing antibodies directed against VIP. The injection of such antibodies [31] induces a decrease in the plasma concentration prolactin and the interruption of brooding, but simultaneously induces a reduction in LH levels in the dwarf hen, which reduction is prejudicial to laying.
An active anti-VIP immunization strategy has also been described in the international patent application published under No. WO 94/08616. However, the results obtained by other authors with this method have been deemed ambiguous and scarcely satisfactory (Sharp, P. J., Sterling, R. J., Talbot, R. T., and Huskinson, N. S., in J. Reprod. Fert., Vol. 11 (1993), pp. 38 and 66), due to the fact that although the VIP unquestionably stimulates the secretion of prolactin, it also has an effect on the secretion of LH (a hormone that is indispensable to the proper progress of reproduction). In two recent articles (in Biol. Reprod., Vol. 50 (1994), pp. 1350-1356, and in Biol. Reprod., Vol. 50 (1994), pp. 1344-1349), the team led by Dr. El Halawani (the inventor in the international patent application cited above) also reported the ambiguity of the role of VIP with regard to the secretion of prolactin.
A link between the variations in prolactinemia and the expression of brooding behavior has been suggested (by Etches et al., in Poult. Sci., Vol. 61, Part 7 (1982), pp. 1354-1362), but no causal relationship has been formally demonstrated. Nor has any determination been made of whether the elevation in prolactinemia is the cause or the consequence of the expression of brooding, or even the simultaneous result of another mechanism. In a recent article (Guemene, D., and Williams, J. B., in Reprod. Nutri. & Develop., Vol. 34 (1994), pp. 371-381) the present inventors also demonstrated that on the contrary, depending on the raising method employed, the turkeys that expressed brooding had lower prolactin levels. It was also shown that high plasma levels of prolactin are not necessary for resumption of the expression of brooding after an interruption due to temporary deprivation of the nest (El Halawani, Burke, and Dennisson, in Biologie et Reproduction, Vol. 23 (1980), pp. 118-123). Thus, the latter demonstration contradicts the hypothesis regarding the causal role of prolactin.
Thus, the experimental results of the various currently available pharmacological approaches do not support or encourage the use of these approaches on breeding farms. Consequently, the physical procedures are still the only type of treatment available to breeders.
The understanding of the hormonal mechanisms associated with the expression of brooding behavior is accompanied by fundamental studies conducted in an attempt to characterize these substances, particularly prolactin [32] [33], and to identify the corresponding genes in various species of birds. Consequently, the prolactin genes for hens [34] [35] and for turkeys [36] [37] have been identified, sequenced, and cloned.
In particular, it has been proposed that the expression vector pGEX-2T [38] be utilized to produce recombinant turkey prolactin [39].
The inventors have made use of the entire above-mentioned body of academic knowledge in order to research new methods that enable the design and manufacture of pharmaceutical compositions that are capable of preventing and/or treating effectively the expression of brooding behavior in birds, and more particularly in the turkey, without inducing either a negative effect on reproduction or the continuation or resumption of laying.
This goal is reached thanks to pharmaceutical compositions that are characterized by the fact that they include, as an active ingredient, either a hybrid substance consisting of a carrier group and a protein that includes at least one antigenic determinant for prolactin, or a microorganism that expresses at its surface or excretes the said hybrid substance, or antibodies that are directed against at least one prolactin antigen site.